Secretory expression of human ScFv against keratin in Pichia pastoris and its effects on cultured keratinocytes.
We have got the humanized antibody with high affinity and specificity against keratin by phage antibody library technology. To improve protein yields and get high affinity and specific anti-keratin antibody, we chose to express it in Pichia pastoris. Anti-keratin ScFv gene from plasmid p3MH/ScFv was subcloned into vector pPIC9K. After confirmed by DNA sequence analysis, the recombinant plasmid pPIC9K/ScFv was transduced into the genome of GS115 P. pastoris. Mut(s) multiple insert transformants were screened by G418 and induced by 5 mL/L methanol to express soluble ScFv. After 6 days of methanol induction, anti-keratin ScFv was efficiently secreted into the medium. Western blot and ELISA assay proved the expressed protein had specific keratin-binding activity. After purification, we examined its effect on cultured keratinocytes by Cell cycle analysis, Which indicated that human ScFv against keratin 17 can inhibit the proliferation of keratinocytes by influence the synthesize of keratinocyte DNA. The successful expression of anti-keratin ScFv in P. pastoris laid a solid foundation for its further application.
Fan
Jianyong
J
Dermatology Center of Chinese PLA, XiJing Hospital, Forth Military Medical University, 710032 Xi'an, China. fanjianyongfmmu@sina.com.cn [corrected]
Shen
Zhu
Z
Wang
Gang
G
Yang
Huilan
H
Liu
Yufeng
Y
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D000915:Antibody Affinity; D001323:Autoantibodies / Q000276:immunology; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D017449:Dermatitis, Allergic Contact / Q000276:immunology* / Q000473:pathology / Q000503:physiopathology / Q000628:therapy; D000939:Epitopes; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007167:Immunotherapy; D053537:Keratin-17 / Q000276:immunology; D015603:Keratinocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D019151:Peptide Library; D010843:Pichia / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011565:Psoriasis / Q000276:immunology* / Q000473:pathology / Q000503:physiopathology / Q000628:therapy; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D007128:Immunoglobulin Fragments; D053537:Keratin-17; D019151:Peptide Library; D011993:Recombinant Fusion Proteins